CN110407951B - Polysaccharide from natural sources and application of polysaccharide in preparation of cosmetics for promoting skin repair and improving skin elasticity - Google Patents
Polysaccharide from natural sources and application of polysaccharide in preparation of cosmetics for promoting skin repair and improving skin elasticity Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
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Abstract
The invention discloses a polysaccharide from natural sources and application of the polysaccharide in promoting skin repair and improving skin elasticity so as to prepare cosmetics, wherein the polysaccharide is prepared by the following method: pulverizing dried whole plant of Petasites japonicus, adding anhydrous ethanol, extracting under reflux, filtering, adding distilled water into the residue, heating, extracting, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain extract, adding appropriate amount of distilled water, stirring for dissolving, filtering, adding 4 times volume of anhydrous ethanol into the filtrate, stirring, precipitating with ethanol, centrifuging, discarding supernatant, washing with acetone and anhydrous ethanol in sequence, dissolving with appropriate amount of distilled water, and lyophilizing. The polysaccharide can promote the proliferation and migration of human skin fibroblasts, has the effect of promoting skin repair, can promote the human skin fibroblasts to generate collagen and elastin, has the effect of improving skin elasticity, and can be used for preparing cosmetics for promoting skin repair and improving skin elasticity.
Description
Technical Field
The invention belongs to the field of cosmetics, relates to application of natural polysaccharide in cosmetics, and particularly relates to application of natural polysaccharide in preparation of cosmetics for promoting skin repair and improving skin elasticity.
Background
Beauty is the nature of a person, but with age, the ability of a person to repair skin and the elasticity of skin decreases.
The skin repair ability of a human is mainly determined by the proliferation rate and migration ability of human skin fibroblasts, and after the human ages, the decrease of the proliferation rate and migration ability of the skin fibroblasts is a direct cause of the decrease of the skin repair ability.
Human skin elasticity is primarily related to collagen and elastin content. Collagen and elastin respectively constitute collagen fiber and elastic fiber, and collagen fiber mainly supports the volume, keeps moisture, lets skin plump, and elastic fiber mainly makes skin can stretch and kick-back, and the elasticity of skin is decided to the two jointly.
Therefore, in order to improve the skin repair ability and skin elasticity of human, it is necessary to find a component which can improve the proliferation rate and migration ability of skin fibroblasts and the synthesis of collagen and elastin.
The polysaccharide from natural plants is widely applied to the field of cosmetics, such as whitening, freckle removing, moisturizing and the like, because the polysaccharide has good compatibility with human skin, is mild, has no stimulation and has sufficient sources.
The petasites japonicus is a very common perennial herb, but the utilization degree is extremely low, so that the great resource waste is caused.
Disclosure of Invention
The invention aims to provide a natural polysaccharide and a second purpose of providing the application of the polysaccharide in promoting skin repair, improving skin elasticity and preparing cosmetics.
The technical scheme for realizing the purpose of the invention is as follows:
a polysaccharide of natural origin, prepared by the following process: pulverizing dried whole plant of Petasites japonicus, adding anhydrous ethanol, extracting under reflux, filtering, adding distilled water into the residue, heating, extracting, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain extract, adding appropriate amount of distilled water, stirring for dissolving, filtering, adding 4 times volume of anhydrous ethanol into the filtrate, stirring, precipitating with ethanol, centrifuging, discarding supernatant, washing with acetone and anhydrous ethanol in sequence, dissolving with appropriate amount of distilled water, and lyophilizing.
Preferably, in the degreasing step, anhydrous ethanol is added according to the feed-liquid ratio of 1:5 for hot reflux extraction for 3h for degreasing.
Preferably, in the step of extracting with distilled water, the filter residue is added with distilled water according to the material-liquid ratio of 1:10, and the mixture is heated and extracted at 85 ℃ for 12 hours.
Preferably, the alcohol precipitation time is 12 h.
Use of the above polysaccharide for promoting skin repair.
The use of the above polysaccharide for the preparation of a cosmetic for promoting skin repair.
Use of the above polysaccharide for increasing skin elasticity.
Use of the above polysaccharide for the preparation of a cosmetic for improving skin elasticity.
The beneficial effects are that:
the natural polysaccharide provided by the invention can promote the proliferation and migration of human skin fibroblasts on one hand, and has the effect of promoting skin repair; on the other hand, the collagen and the elastin can be promoted to be produced by human skin fibroblasts, and the effect of improving the skin elasticity is achieved. Moreover, the polysaccharide has no obvious stimulation to human skin, so the polysaccharide has the value of preparing cosmetics for promoting skin repair and improving skin elasticity.
Drawings
FIG. 1 is a photograph taken with an inverted microscope in a cell scratch test;
FIG. 2 shows the Western blot results of the expression levels of type I collagen and elastin.
Detailed Description
First, experiment method
1. Preparation of polysaccharides
Pulverizing dry whole plant of Petasites japonicus, adding anhydrous ethanol according to a material-liquid ratio of 1:5(kg: L), performing hot reflux extraction for 3h for degreasing, repeatedly degreasing for 1 time, filtering, adding distilled water into filter residue according to a material-liquid ratio of 1:10(kg: L), heating and extracting at 85 ℃ for 12h, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain an extract, adding distilled water according to a material-liquid ratio of 1:5(kg: L), stirring to fully dissolve, performing suction filtration, adding 4 times of volume of anhydrous ethanol into the filtrate, stirring uniformly, performing alcohol precipitation for 12h, centrifuging, discarding supernatant, washing with acetone and anhydrous ethanol in sequence, dissolving with an appropriate amount of distilled water, and freeze-drying to obtain Petasites japonicus polysaccharide.
2. Human skin fibroblast cell culture and passage
Human skin fibroblast HFF-1 cells were cultured in DMEM containing 15% fetal bovine serum and 1% double antibody at 37 deg.C and 5% CO2Culturing in incubator, changing culture solution every 3d, digesting with 0.25% trypsin when cell growth fusion reaches about 85%, and subculturing at 1:3 ratio. Cells in the logarithmic growth phase were taken for the experiment.
3. Skin repair promoting Activity test
3.1 promotion of human skin fibroblast proliferation (MTT test)
Collecting cells in logarithmic growth phase, digesting, and preparing into 1 × 10 concentration DMEM medium containing 15% fetal calf serum and 1% double antibody5The cell suspension was inoculated in a 96-well plate at 150. mu.L/well, 37 ℃ and 5% CO2After culturing in an incubator for 24h, changing the culture medium to DMEM culture medium containing 50 mug/mL petasites polysaccharide, 15% fetal calf serum and 1% double antibody for continuous culture, after continuously culturing for 24h and 48h, removing the culture medium by suction, washing with PBS, adding 150 muL DMEM culture medium containing 15% fetal calf serum and 1% double antibody and 20 muL LMTT solution (5mg/mL), 37 ℃, 5% CO2Culturing for 4h, removing supernatant, adding DMSO 150 μ L into each well, and shaking for 10min to dissolve the crystals. Meanwhile, a control group is arranged, compared with a polysaccharide group, the control group is not added with the butterbur polysaccharide, the rest is completely the same, and each group has 5 multiple holes. The absorbance of each well was measured at 490nm, and the proliferation promoting rate of the polysaccharide group was calculated according to the calculation formula shown below.
Proliferation-promoting ratio (%) - (absorbance value of polysaccharide group-absorbance value of control group) ÷ absorbance value of control group × 100%
3.2 promotion of human skin fibroblast migration (cell scratch test)
Collecting cells in logarithmic growth phase, digesting, and preparing into 1 × 10 concentration DMEM medium containing 15% fetal calf serum and 1% double antibody5The cell suspension was inoculated in 6-well plates at 2 mL/well, 37 ℃ and 5% CO2After 24h of culture in an incubator, the center of each culture well is scratched straight with the tip of a 1mL pipette gently, PBS is washed, 2mL of DMEM medium containing 50 mug/mL of Petasites, 15% of fetal bovine serum and 1% of double antibody is added to the polysaccharide group, 2mL of DMEM medium containing 15% of fetal bovine serum and 1% of double antibody is added to the control group, 3 multiple wells are formed, the culture is continued for 24h and then observed and photographed under an inverted microscope, the scratch distance is measured by using Image J software, and the mobility promotion rate of the polysaccharide group is calculated according to the calculation formula shown below.
Migration rate (%) - (control scratch distance-polysaccharide scratch distance) ÷ control scratch distance × 100%
4. Test for improving skin elasticity activity
The effect of the Petasites japonicus polysaccharide on the expression level of type I collagen and elastin in human skin fibroblasts was determined by a Westernblot method. The specific method comprises the following steps: collecting cells in logarithmic growth phase, digesting, and preparing into 1 × 10 concentration DMEM medium containing 15% fetal calf serum and 1% double antibody5The cell suspension was inoculated in 6-well plates at 2 mL/well, 37 ℃ and 5% CO2After culturing for 24 hours in an incubator, a polysaccharide group replacement culture medium is a DMEM culture medium containing 20, 50 mu g/mL of petasites japonicus polysaccharide, 15% fetal bovine serum and 1% double antibody, a control group replacement culture medium is a DMEM culture medium containing 15% fetal bovine serum and 1% double antibody, continuously culturing for 48 hours, sucking the culture medium, washing with PBS, collecting cells, cracking, extracting total protein according to BCA kit instructions and calculating the protein concentration, respectively taking 50 mu g of protein to perform SDS-PAGE electrophoresis separation, transferring the protein to a PVDF membrane, sealing with skim milk powder, incubating overnight at 4 ℃ under a primary antibody, washing, adding a secondary antibody, incubating for 1 hour at room temperature, and photographing by a chemiluminescence imaging system to compare the difference of the collagen I expression levels and the elastin expression levels of the polysaccharide group and the control group.
5. Skin irritation test
According to technical Specification for safety of cosmetics (2015), 30 test persons are selected according to the selection standard of a test subject, qualified patch materials are selected, 0.025g of the Petasites japonicus polysaccharide prepared by the method is put into a patch tester, and no substance is added into a control hole. The spot tester with the tested object is pasted on the back of a volunteer by using a non-irritating adhesive tape, the spot tester is lightly pressed by hands and palms to be uniformly pasted on the skin, and the spot tester is continuously kept on the skin for 24 hours. The interval is 30min after the plaque remover is removed, and the skin reaction is observed after the indentation disappears. The observation was done 24 and 48h after the patch test. And judging the result, and reacting at the 0 stage: no reaction occurs at the tested part; stage 1 reaction: pale erythema on the skin; 2, stage reaction: erythema, infiltration, or pimples of the skin; 3, stage reaction: edematous erythema, papules, etc. of the skin; 4, stage reaction: the skin appeared noticeably red, swollen, with papules or bullae.
6. Statistical treatment
Using SPSS17.0 analysis, data are presented as mean ± standard deviation, homogeneity of variance is tested using One-way ANOVA with One-way analysis of variance, and analyzed with Student's-t test, P <0.05 indicating that the difference is significant.
Third, experimental results
1. Polysaccharide promoting skin repair activity
The MTT test results are shown in Table 1, compared with the control group, the absorbance value of the polysaccharide group is obviously increased, and the polysaccharide has time dependence on the proliferation promoting rate of human skin fibroblasts.
TABLE 1 Absorbance values for each set at different time points
| Control group | Polysaccharide group | Proliferation promoting rate | |
| 24h | 0.36±0.09 | 0.55±0.11 | 52.8％ |
| 48h | 0.58±0.12 | 0.97±0.14 | 67.2％ |
As shown in fig. 1, the scratch distance of the polysaccharide group was significantly reduced compared to the control group, and the migration promoting rate of the petasites japonicus polysaccharide was (73.9 ± 3.4)% (composite wells 1: 71.9%, 2: 71.9%, and 3: 77.8%).
MTT test results and cell scratch test results show that the butterbur polysaccharide can promote the proliferation and migration of human skin fibroblasts and has the effect of promoting skin repair.
2. Polysaccharide for improving skin elasticity
The result of the Western blot method is shown in figure 2, compared with the control group, the polysaccharide group has the advantages that the expression level of the type I collagen and the elastin of the human skin fibroblasts is obviously increased, and the concentration dependency is achieved.
3. Test results of polysaccharide Patch
The patch test results are shown in table 2, and it can be seen that the butterbur polysaccharide is mild to human skin, and can be used for preparing cosmetics to improve the effects of the cosmetics in promoting skin repair and improving skin elasticity.
TABLE 2 Patch test results
The above examples show that the natural polysaccharide provided by the invention can promote the proliferation and migration of human skin fibroblasts, and has the effect of promoting skin repair; on the other hand, the collagen and the elastin can be promoted to be produced by human skin fibroblasts, and the effect of improving the skin elasticity is achieved. Moreover, the polysaccharide has no obvious stimulation to human skin, so the polysaccharide has the value of preparing cosmetics for promoting skin repair and improving skin elasticity.
Claims (1)
1. Use of a polysaccharide of natural origin for the preparation of a cosmetic product for promoting skin repair or improving skin elasticity, the polysaccharide being prepared by: crushing the dried whole herb of the petasites japonicus, adding absolute ethyl alcohol according to the material-liquid ratio of 1kg to 5L, carrying out hot reflux extraction for 3h for degreasing, repeatedly carrying out degreasing for 1 time, filtering, adding distilled water into filter residues according to the material-liquid ratio of 1kg to 10L, heating and extracting at 85 ℃ for 12h, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain an extract, adding distilled water according to the material-liquid ratio of 1kg to 5L, stirring for full dissolution, carrying out suction filtration, adding 4 times of volume of absolute ethyl alcohol into the filtrate, stirring uniformly, carrying out alcohol precipitation for 12h, centrifuging, discarding supernatant, washing with acetone and absolute ethyl alcohol in sequence, dissolving with an appropriate amount of distilled water, and freeze-drying to obtain the petasites.
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| CN114773497B (en) * | 2022-05-16 | 2023-04-28 | 广东雁盟智慧健康科技有限公司 | Rose polysaccharide and application thereof in promoting skin regeneration and repair, increasing skin elasticity and resisting skin aging |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103798672A (en) * | 2014-03-11 | 2014-05-21 | 福建师范大学 | Preparation method of instant arisaema japonicum |
| CN108348564A (en) * | 2015-10-22 | 2018-07-31 | 默克专利股份有限公司 | Method for preparing specific extraction object from comfrey platymiscium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103798672A (en) * | 2014-03-11 | 2014-05-21 | 福建师范大学 | Preparation method of instant arisaema japonicum |
| CN108348564A (en) * | 2015-10-22 | 2018-07-31 | 默克专利股份有限公司 | Method for preparing specific extraction object from comfrey platymiscium |
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| Title |
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| "ДИНАМИКА НАКОПЛЕНИЯ ВОДОРАСТВОРИМЫХ ПОЛИСАХАРИДОВ";О.Г. Зорикова等;《ХИМИЯ РАСТИТЕЛЬНОГО СЫРЬЯ》;20151231(第4期);第155-158页 * |
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